Our goal is to understand the mechanisms that control the timing of the initiation of DNA replication as a function of the cell cycle. We propose to study this crucial event in Caulobacter crescentus, an organism whose developmental cell cycle exhibits inherent asymmetry. The newly replicated chromosomes differ in replication potential: the chromosome that partitions to the progeny stalked cell immediately initiates DNA replication whereas the progeny swarmer cell chromosome does not initiate replication until later in the cell cycle. Caulobacter is uniquely suited to a study of the factors that control DNA replication because it has a single chromosome that initiates replication from an identified origin once per cell cycle, in a cell type that can be easily obtained from synchronized populations. We have recently isolated mini-chromosomes driven solely by the cloned origin that correctly initiate replication coincident with the bona fide chromosomal origin. The proposed research has four main objectives. The first is to define the replication origin sequences and cognate factors that mediate the differential initiation of DNA replication. To do this we will identify, both in vivo and in vitro, factors that bind to regions of the origin that are essential for replication initiation or for the timing of replication. We will also determine if a promoter, shown to reside within the origin and to be selectively expressed from replication-competent chromosome, contributes to the control of replication initiation. We will also attempt to directly visualize origin-specific transcripts at one pole of the predivisional cell using in situ hybridization. The second objective is to isolate and characterize mutants defective in DNA replication and/or chromosome segregation. The third is to determine the mechanisms that control the cell cycle expression of identified enzymes and factors that are involved in DNA replication, such as DnaA, the beta subunit of DNA polymerase III, and gyrase. The fourth objective is to determine the mechanisms that regulate the cell cycle expression of a newly identified DNA methyltransferase, and examine the role of this methylation system in the control of replication initiation and cell differentiation. These studies will address the underlying mechanisms that control polarity during the bacterial cell cycle.